Estrogens, acting via estrogen receptor ? (ER?), were known to regulate gene expression and to activate signal transduction pathways. We identified a conserved extranuclear pathway by which 17?-estradiol (E2), acting through ER?, rapidly activates phosphoplipase C ? (PLC?) leading to production of inositol triphosphate (IP3). The IP3 binds to and opens endoplasmic reticulum (EnR) IP3 receptors (IP3R) leading to extremely rapid (<1 min.) efflux of calcium (Ca2+) from the lumen of the EnR into the cell body. Elevated intracellular Ca2+ primes cells for subsequent actions of E2-ER?; depletion of EnR Ca2+ activates the unfolded protein response (UPR), inducing the important chaperone BiP/GRP78 (glucose regulated protein 78 kDa). Activation of this pathway is required for E2-ER?-regulated gene expression, induction of cell proliferation and protects cells against stress. We target this pathway with our medically promising ER? biomodulator, BHPI, which uses the same pathway as E2, but induces toxic hyperactivation of the UPR. Our hypothesis is that the products of activation of this newly unveiled pathway, elevated intracellular calcium (Aim 1), and at later times, BiP chaperone (Aim 2), link to and regulate subsequent E2-ER?-regulated gene expression and stabilize ER?, influencing drug resistance and genomic actions of ER?. Our goals are to identify the mechanism(s) by which these products couple to, and control, gene expression (Aim 1), ER? stability and response to drugs (Aim 2), and to identify the sensors and signals that allow E2-ER? to rapidly initiate the pathway (Aim 3). Aim 1. Identify the mechanism(s) by which the product of E2-ER? activation of the pathway couples to and controls E2-ER?-regulated gene expression. Test the data-driven hypothesis that Ca2+ produced by pathway activation acts through the Ca2+ sensor calmodulin (CaM) to regulate nuclear E2-ER?:CaM interaction, E2-ER? dimerization and nuclear localization and thereby controls E2-ER?-regulated gene expression. Aim 2. Background: In CRISPR/Cas9 generated cell lines expressing constitutively active ER? mutants, the UPR is activated and ER? is partially resistant to antagonists. Identify the mechanism by which UPR activation contributes to drug resistance. Test the hypothesis that drug resistance in these cells arises in part because ER?, together with progesterone-PR, synergistically activate the UPR, inducing BiP chaperone, which stabilizes ER?, thereby contributing to drug resistant gene expression. Aim 3. Identify components of the multiprotein complex by which E2-ER? initiates the pathway. Using an unbiased CRISPR/Cas9 lethality screen, followed by verification and analysis of multiprotein complexes, we will identify the activating kinase(s), scaffolding proteins, other components of the complex(es), genes that impact the pathway and probe ER? interactions in the complex. These studies will establish the initial events that occur when estrogen contacts a cell and identify new mechanisms coupling steroid receptor regulated transcription to extranuclear signals.